Periventricular leukomalacia (PVL) is the principal lesion underlying cerebral palsy in premature infants, and a key cell-type injured in this lesion is the oligodendrocyte. For this reason, it is important to understand the mechanisms of death present in oligodendrocytes that might be activated in PVL. We and others have developed methods for growing oligodendrocytes in relatively pure culture based on immunopanning and serum-free defined medium. Using these cultures we have found that pre-oligodendrocytes and immature oligodendrocytes are more vulnerable than mature oligodendrocytes to oxidative stress caused by depletion of intracellular glutathione. This injury is blocked by free radical scavangers and is accompanied by intracellular oxygen free radical accumulation. Recent work suggests a critical involvement of 12- lipoxygenase in the pathway leading from depletion of intracellular glutathione to cell death. In addition, preliminary studies of this project and Project 2, in addition to studies by other investigators, suggest that the expression of antioxidant enzymes may be up-regulated in mature OLs. The overall hypothesis is that there are specific properties of pre- oligodendrocytes and immature OLS that contribute to their enhanced sensitivity to oxidative insults; these include an under-expression of antioxidant enzymes and increased activity of 12-lipoxygenase. Our long- term objective is to understand the sequence of events that lead from glutathione depletion and oxidative stress to the death of pre- oligodendrocytes and immature oligodendrocytes, as well as the basis for the resistance of mature oligodendrocytes to this form of injury. The specific aims are to determine: 1. the sensitivity of oligodendrocytes at specific stages of development to a variety of sources for oxidative stress; 2. whether the decline in vulnerability to oxidative stress with developmental progression in the oligodendrocyte lineage is due to up- regulation of one or more antioxidant enzymes; and 3, whether activation of 12-lipoxygenase activity is required for oxidative stress induced oligodendrocyte toxicity. Through this work we will gain fundamental information about how development alters the vulnerability of oligodendrocytes to oxidative stress.